The organelle that most typifies a synapse is the synaptic vesicle. We and others have shown that synaptic vesicles have unique integral and peripheral membrane proteins. Our data suggest that synaptic vesicles owe their unique composition and size to a neuronal specific modification of an endosomal pathway. We plan to identify the hierarchy of sorting signals that sends an integral membrane protein to the endosome and to the synaptic vesicle. We will use two proteins of contrasting membrane topology, synaptophysin and synaptobrevin. If synaptic vesicles turn out to be related developmentally to the transcytotic vesicles of epithelial cells, we will be able to short-cut the search for sorting information by drawing on the wealth of data available for the transcytotic vesicle. To help understand the association of the tyrosine protein kinase pp60(c-src) with synaptic vesicles, we will identify its synaptic vesicle targeting mechanism. Cytosolic proteins involved in the sorting of synaptic vesicle proteins and in generating their unique size will be analyzed using a recently developed in vitro reconstitution system from the neuroendocrine cell line, PC12. Association of synaptic vesicles with the subcortical cytoskeleton will also be examined in semi-intact PC12 cells. Our data will tell us what regions of synaptic vesicle proteins are required for their targeting, and what cytosolic factors are necessary for assembling these proteins into synaptic vesicles.